U.S. patent application number 13/126109 was filed with the patent office on 2012-01-26 for microbial enzymes as detergent additives.
Invention is credited to Shaon Ray Chaudhuri.
Application Number | 20120021489 13/126109 |
Document ID | / |
Family ID | 45066239 |
Filed Date | 2012-01-26 |
United States Patent
Application |
20120021489 |
Kind Code |
A1 |
Chaudhuri; Shaon Ray |
January 26, 2012 |
MICROBIAL ENZYMES AS DETERGENT ADDITIVES
Abstract
Provided in part herein are compositions that include isolated
microbial enzymes, such as amylases, lipases, and proteases that
are useful as detergent additives. Also provided are methods of
isolating amylases, lipases, and proteases from microbes, as well
as methods of using these enzymes as detergent additives, and for
stain removal.
Inventors: |
Chaudhuri; Shaon Ray;
(Kolkata, IN) |
Family ID: |
45066239 |
Appl. No.: |
13/126109 |
Filed: |
July 24, 2010 |
PCT Filed: |
July 24, 2010 |
PCT NO: |
PCT/IB2010/001816 |
371 Date: |
April 26, 2011 |
Current U.S.
Class: |
435/202 ;
435/263; 435/264; 510/392; 510/530 |
Current CPC
Class: |
C12N 9/2417 20130101;
C11D 3/38681 20130101; C12N 9/20 20130101; C12N 9/52 20130101; C11D
3/386 20130101 |
Class at
Publication: |
435/202 ;
435/264; 510/392; 435/263; 510/530 |
International
Class: |
C12N 9/28 20060101
C12N009/28; C12S 11/00 20060101 C12S011/00; C11D 3/386 20060101
C11D003/386; C12S 9/00 20060101 C12S009/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 1, 2010 |
IN |
599/KOL/2010 |
Claims
1. A composition, comprising a Bacillus strain amylase, wherein: a.
said strain comprises a 16SrDNA sequence comprising SEQ ID NO:3; or
b. representative bacteria of said strain have been deposited with
the Microbial Type Culture Collection and designated by deposit
accession number 5567.
2. An enzymatic detergent additive comprising the composition of
claim 1.
3-4. (canceled)
5. A detergent composition comprising an enzymatic detergent
additive of claim 2 and a surfactant.
6. The additive of claim 2, further comprising a lipase.
7. The additive of claim 6, wherein a. the lipase is an
Acinetobacter strain lipase; and b. said strain comprises a 16SrDNA
sequence comprising SEQ ID NO: 2; or c. representative bacteria of
said strain have been deposited with the Microbial Type Culture
Collection and designated by deposit accession number MTCC5563.
8. The additive of claim 2, further comprising a protease.
9. The additive of claim 8, wherein a. the protease is a
Microbacterium strain protease; and b. said strain comprises a
16SrDNA sequence comprising SEQ ID NO:1; or c. representative
bacteria of said strain have been deposited with the Microbial Type
Culture Collection and designated by deposit accession number
MTCC5565.
10. The additive of claim 8, further comprising a lipase.
11. The additive of claim 9, further comprising a lipase, wherein
a. the lipase is an Acinetobacter strain lipase; and b. said strain
comprises a 16SrDNA sequence comprising SEQ ID NO: 2; or c.
representative bacteria of said strain have been deposited with the
Microbial Type Culture Collection and designated by deposit
accession number MTCC5563.
12. A detergent composition comprising an enzymatic detergent
additive of claim 7, and a surfactant.
13-14. (canceled)
15. A process for production of an amylase, comprising: a.
culturing a Bacillus strain in growth medium; b. removing cell-free
cultured media from the culture; and c. purifying the amylase from
the cell-free culture media; wherein the Bacillus strain comprises
a 16SrDNA sequence comprising SEQ ID NO: 3; or wherein
representative bacteria of the Bacillus strain have been deposited
with the Microbial Type Culture Collection and designated by
deposit accession number MTCC5567.
16. The process of claim 15, wherein the Bacillus strain is
cultured under batch fermentation conditions.
17. The process of claim 15, wherein the Bacillus strain is
immobilized.
18. A process for removing a stain from a surface, comprising
contacting said stain with the composition of claim 1.
19. The process of claim 18, wherein said surface is selected from
the group consisting of fabric, flooring, ceramic, glass, or
metal.
20. The process of claim 18, further comprising contacting said
stain with a lipase.
21. The process of claim 20, wherein a. the lipase is an
Acinetobacter strain lipase; and b. said strain comprises a 16SrDNA
sequence comprising SEQ ID NO: 2; or c. representative bacteria of
said strain have been deposited with the Microbial Type Culture
Collection and designated by deposit accession number MTCC5563.
22. The process of claim 18, further comprising contacting said
stain with a protease.
23. The process of claim 22, wherein a. the protease is a
Microbacterium strain protease; and b. said strain comprises a
16SrDNA sequence comprising SEQ ID NO:1; or c. representative
bacteria of said strain have been deposited with the Microbial Type
Culture Collection and designated by deposit accession number
MTCC5565.
24. The process of claim 23, further comprising contacting said
stain with a lipase, wherein a. the lipase is an Acinetobacter
strain lipase; and b. said strain comprises a 16SrDNA sequence
comprising SEQ ID NO: 2; or c. representative bacteria of said
strain have been deposited with the Microbial Type Culture
Collection and designated by deposit accession number MTCC5563.
Description
TECHNICAL FIELD
[0001] The technology relates in part to isolated microbial
amylases, lipases, and proteases suitable for use as detergent
additives. The technology also relates in part to methods for
removing stains using isolated microbial enzymes.
BACKGROUND
[0002] Detergents are used for cleaning various types of fabrics
and hard surfaces. Enzymes, such as protease, amylase, and lipase
may be used as detergent additives to improve cleaning efficiency.
Protease hydrolyzes proteins into soluble amino acids. Amylase
catalyzes the break down of starch-based stains into smaller
segments of oligosaccharides and dextrins, which are water soluble.
Lipase hydrolyzes triglycerides into mono and diglycerides,
glycerol, and free fatty acids, which are more soluble than fats.
These more soluble reaction products may be more easily removed
from fabrics and surfaces, increasing the cleaning efficiency of
the detergent used.
[0003] Natural microbial isolates may be used to produce enzymes,
and these natural sources may be obtained from different
environments. Urban areas sometimes dispose of waste in wetlands,
and environmental samples from these waste dumps may contain a
richly bio-diverse source of microbes.
SUMMARY
[0004] Provided herein are compositions that include isolated
microbial enzymes, such as amylases, lipases, and proteases that
are useful as detergent additives. Also provided are methods of
isolating amylases, lipases, and proteases from microbes, as well
as methods of using these enzymes as detergent additives, and for
stain removal.
[0005] Thus featured in some embodiments are compositions including
a Bacillus strain amylase, where: the strain includes a 16SrDNA
sequence including SEQ ID NO: 3; or representative bacteria of the
strain have been deposited with the Microbial Type Culture
Collection and designated by deposit accession number MTCC5567.
Also provided are detergent compositions that include the enzymatic
detergent additive and a surfactant, in certain embodiments.
[0006] In some embodiments, also provided are enzymatic detergent
additives that include the amylase. In some embodiments, an
additive is in the form of a granulate, a powder, or a liquid, and
in some embodiments, the additive further includes an enzyme
stabilizer. In some embodiments, the additive further includes a
surfactant.
[0007] In some embodiments, also provided are additives including
an amylase and further including a lipase. In some embodiments, the
protease is an Acinetobacter strain protease; and the strain
includes a 16SrDNA sequence including SEQ ID NO: 2; or
representative bacteria of the strain have been deposited with the
Microbial Type Culture Collection and designated by deposit
accession number MTCC5563
[0008] In some embodiments, also provided are additives including
an amylase and further including a protease. In some embodiments,
the protease is a Microbacterium strain protease; and the strain
includes a 16SrDNA sequence including SEQ ID NO: 1; or
representative bacteria of the strain have been deposited with the
Microbial Type Culture Collection and designated by deposit
accession number MTCC5565
[0009] Also provided are enzymatic detergent additives that include
an amylase and a lipase, additives that include an amylase and a
protease, and additives that include an amylase, a protease, and a
lipase, in certain embodiments. In some embodiments, the enzymatic
detergent additive further includes a surfactant. In some
embodiments the enzymatic detergent additive further includes an
enzyme stabilizer. In some embodiments, the enzymatic detergent
additive is in the form of a granulate, a powder, or a liquid.
[0010] Also featured in some embodiments is a process for
production of an amylase, including culturing a Bacillus strain in
growth medium; removing cell-free cultured media from the culture;
and purifying the amylase from the cell-free culture media; where
the Bacillus strain includes a 16SrDNA sequence including SEQ ID
NO: 3; or where representative bacteria of the Bacillus strain have
been deposited with the Microbial Type Culture Collection and
designated by deposit accession number MTCC5567. In some
embodiments, the Bacillus strain is cultured under batch
fermentation conditions, and sometimes the Bacillus strain is
immobilized.
[0011] Also provided in some embodiments is a process for removing
a stain from a surface, including contacting the stain with a
Bacillus strain amylase herein. In some embodiments, the surface is
selected from the group consisting of fabric, flooring, ceramic,
glass, or metal. In some embodiments, the process further includes
contacting the stain with a lipase, including contacting the stain
with a lipase described herein. In certain embodiments, the process
further includes contacting the stain with a protease, including
contacting the stain with a described herein. In some embodiments,
the process further includes contacting the stain with a lipase and
with a protease, including contacting the stain with an
Acinetobacter strain lipase or a Microbacterium strain protease
described herein.
[0012] The foregoing summary illustrates certain embodiments and
does not limit the disclosed technology. In addition to
illustrative aspects, embodiments and features described above,
further aspects, embodiments, and features will become apparent by
reference to the drawings and the following detailed description
and examples.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] The drawings illustrate embodiments of the technology and
are not limiting. For clarity and ease of illustration, the
drawings are not made to scale and, in some instances, various
aspects may be shown exaggerated or enlarged to facilitate an
understanding of particular embodiments.
[0014] FIG. 1a is a graph representing a pH profile of the isolated
protease enzyme from SRC_GZ. FIG. 1b is a graph representing a
thermostability profile of the isolated protease enzyme from
SRC_GZ.
[0015] FIG. 2 is a graph representing a wash performance analysis
of stain intensity, with and without addition of protease to
detergent.
[0016] FIG. 3 is a graph representing a wash performance analysis
of stain density, with and without addition of protease to
different detergents.
[0017] FIG. 4 is a graph representing a wash performance analysis
of various fabrics, with and without addition of protease to
detergent.
[0018] FIG. 5 is a graph representing a wash performance analysis
of various types of stains, with and without addition of protease
to detergent.
[0019] FIG. 6a is a graph representing an analysis of the shelf
life of a protease additive and detergent mixture.
[0020] FIG. 6b is a photograph showing an analysis of the shelf
life of a protease additive and detergent mixture.
[0021] FIG. 7 is a graph representing an analysis of protease
activity obtained from immobilized cells over time.
[0022] FIG. 8a-8f are charts representing market survey results
depicting the acceptability of a detergent formulation including
protease and lipase additives where the darker shade depicts the
percentage of activity.
[0023] FIG. 9 is a photograph showing amylase activity on a
starch-containing plate.
[0024] FIG. 10 is a photograph showing a wash performance analysis
of stained cloth treated with protease, lipase, and amylase as
detergent additives. The enzymes were produced using packed bed
reactors.
DETAILED DESCRIPTION
[0025] In the following detailed description, reference is made to
the accompanying drawings, which form a part hereof. In the
drawings, similar symbols typically identify similar components,
unless context dictates otherwise. Illustrative embodiments
described in the detailed description, drawings, and claims do not
limit the technology. Other embodiments may be utilized, and other
changes may be made, without departing from the spirit or scope of
the subject matter presented herein. It will be readily understood
that aspects of the present disclosure, as generally described
herein, and illustrated in the drawings, can be arranged,
substituted, combined, separated, and designed in a wide variety of
different configurations, all of which are explicitly contemplated
herein.
[0026] Enzymes may be used to improve the cleaning efficiency of
detergents, including, for example, liquid, powder, and granular
detergents. The performance of enzymes in detergents depends on a
number of factors, such as, for example, the composition of the
detergent, type of stains to be removed, wash temperature, washing
procedure and wash-water hardness. Besides high temperature and
alkalinity, the enzyme often must withstand the presence of
detergent additives such as, for example, bleaching agents, bleach
activators, surfactants, perfumes, and the like. Microbe-produced
proteases can be used as detergent additives (Ganesh Kumar, C. and
H. Tyagi, 1999. Microbial alkaline protease: From a bioindustrial
viewpoint. Biotechnol. Adv., 17: 561-594). These enzymes hydrolyze
protein-based stains in fabrics into soluble amino acids. Among the
various proteases, bacterial proteases often are utilized and
sometimes animal or fungal proteases are used. Bacterial proteases
selected often are extracellular, readily produced in large amount
and are often thermostable and active at wider pH ranges. One
factor to be considered during the selection of the enzyme is its
stability and the shelf life of the enzymatic action. For
industrial applications, the immobilization of enzyme on a solid
support can offer additional advantages like repeated use of the
enzyme, ease of product separation and improvement in stability
(Ahmed, S. A., et al., 2007. Stabilization of Bacillus
licheniformis ATCC21415 alkaline protease by immobilization and
modification. Aust. J. Basic Applied Sci., 1 (3): 313-322).
[0027] Other enzymatic detergent additives are amylase, cellulose,
and lipase. Enzymes such as amylases, proteases and lipases can
improve washing efficiency. Amylase catalyzes the break down of
starch based stains into smaller segments of oligosaccharides and
dextrins which are water soluble. Lipase can be used in detergent
formulations to remove fat-containing stains such as those
resulting from frying fats, butter, salad oils etc. The enzyme
hydrolyzes triglycerides into mono and diglycerides, glycerol and
free fatty acids which are more soluble than fats. Protease
catalyzes the breakdown of proteins into small peptides and amino
acids.
[0028] Because they are often active under moderate conditions,
such as warm temperatures and neutral pH, they may reduce energy
consumption by eliminating the need to maintain extreme
environments, as required by many chemically catalyzed reactions.
The reaction specificities of the enzymes minimize the amount of
by-products, and therefore may offer a lower risk to the
environment than chemically catalyzed reactions.
[0029] In certain embodiments, microbial enzymes, such as protease,
lipase, and amylase, are isolated from bacteria. These enzymes,
alone, or in combination, may be used as detergent additives to
enhance stain removal.
[0030] By "isolated" is meant that the enzyme, protein,
polypeptide, or peptide, is separated from other cellular
materials. For example, a protein may be isolated from bacteria by
being secreted into a supernatant, and the supernatant then
separated from the protein-producing bacteria. Or, for example, the
protein may be further purified using additional separation
techniques that would separate the protein from some other
materials in the supernatant.
[0031] The term "protease" includes all proteins, polypeptides, and
peptides having a protease activity, including a peptidase and/or a
proteinase activity. Proteases can catalyze the hydrolysis of
peptide bonds. The term "Microbacterium strain protease" includes
proteases of Microbacterium origin, and may be cellular, secreted,
or isolated. The protease may be isolated from cell culture using
standard protease isolation methods, and the protease
characteristics discussed herein.
[0032] The term "lipase" includes all proteins, polypeptides, and
peptides having a lipase activity. Lipases catalyze the hydrolysis
of ester bonds in water-insoluble, lipid substrates. The term
"Acinetobacter strain lipase" includes lipases of Acinetobacter
origin and may be cellular, secreted, or isolated. The lipase may
be isolated from cell culture using standard lipase isolation
methods, and the lipase characteristics discussed herein.
[0033] The term "amylase" includes all proteins, polypeptides, and
peptides having an amylase activity. Amylases catalyze the
breakdown of starch, such as carbohydrates, into sugars. The term
"Bacillus strain amylase" includes amylases of Bacillus origin and
may be cellular, secreted, or isolated. The amylase may be isolated
from cell culture using standard amylase isolation methods, and the
amylase characteristics discussed herein.
[0034] An article may be contacted with a detergent, additive or
detergent-additive for stain removal. By "contacting" is meant that
the detergent, additive, or detergent-additive formulation is
brought into close contact with, including touching, a surface,
such as a solid or fabric surface, such as, for example, fabric,
flooring, ceramic, glass, or metal.
[0035] For example, the enzymes may be present in the composition
that contacts the surface at levels from about 0.00001% to about 2%
enzyme protein by weight of the composition.
[0036] In other examples, the enzymes may be present in proportions
of enzyme to detergent at levels from about 3 units to about 7
units enzyme per gram detergent.
[0037] Enzyme additive formulations include enzymes that are, for
example, from about 80% to about 90% free of other proteins. Where
enzyme additives include more than one enzyme, the other proteins
include those that are not one of the enzymes. For example, where
an enzyme additive includes protease and lipase, the "other
proteins" are those that are not protease or lipase.
[0038] In certain embodiments, the detergent and enzymatic
detergent additive compositions include one or more enzyme
stabilizers, surfactants, and/or fillers. An "enzymatic detergent
additive" may include one or more enzymes selected from the group
consisting of proteases, lipases, and amylases. The enzymatic
detergent additive may be, for example, formulated as a powder,
granulate, or a liquid. By "enzyme stabilizer" is meant a
composition that increases the storage stability of an enzyme.
Enzyme stabilizers may include, for example, propylene glycol,
calcium chloride, glycine. By "surfactant" is meant a surface
active agent, including, for example, anionic, nonionic,
ampholytic, cationic, and zwitterionic surfactants, and mixtures
thereof. By "filler" is meant a molecule or compound that is used
to dilute or distribute the active ingredients of the detergent
composition, in appropriate concentrations. Fillers may include,
but are not limited to, sodium sulphate for powder cleaning
compositions, and water for liquid cleaning compositions.
[0039] According to certain embodiments, the enzymatic detergent
additives may be used with or incorporated into a detergent
composition, such as, but not limited to, a fabric care
composition, a dish cleaning composition, and a home care
composition. Examples of fabric care compositions include, but are
not limited to, liquid laundry detergents, solid laundry
detergents, powder laundry detergents, granular laundry detergents,
laundry soap products, heavy duty liquid detergents, rinse
additives, laundry spray treatment products, and laundry
pre-treatment products. Examples of dish cleaning compositions
include, but are not limited to, hand dish washing detergents,
solid granular dish soap, and automatic dish washing detergents.
Examples of home care compositions include, but are not limited to,
hard surface cleaning detergents, floor cleaning compositions,
window cleaning compositions, toilet and bathroom cleaning
compositions, car washing detergents, and rug or carpet cleaning
detergents. The detergent compositions may be, for example, in
granular, powder, liquid, gel, paste, bar, or flake form.
[0040] Certain proteases, amylases and lipases useful for preparing
detergent additives are described in further detail hereafter.
Proteases
[0041] A particular protease enzyme was isolated from a bacterial
culture (SRC_GZ) obtained from an old dumping ground [22.degree.32'
17.5'' N and 88.degree.23' 53.7''E (using GOOGLE EARTH)] at East
Calcutta Wetland, that has been converted into a recreational
centre with a forest like vegetation It is a gram negative bacillus
that produces protease and catalase but does not secrete DNAse,
lipase, oxidase, lecithinase or amylase. The molecular
identification of the isolate was based on a partial sequence
analysis of 16SrDNA, and the sequence is available at GenBank as
Acinetobacterium SRC-010, with the accession number E0006695. This
partial sequence shows 98.22% identity with Microbacterium
luteolum. The bacteria is resistant to Polymyxin B (100 .mu.g),
Cloxacillin (30 .mu.g), Rifampicin (15 .mu.g), Cephotaxime (30
.mu.g), Metronidazole (4 .mu.g), Trimethoprin (30 .mu.g) and
Vancomycin (30 .mu.g) as per the protocol of Himedia Laboratories
Pvt Ltd. The sensitivity of the strains against different
antibiotics was determined by disk diffusion method using
commercially available (Himedia) antibiotic discs. The
representative of each group of antibiotic had been tasted. The
antibiotics used were Ampicillin (A10), Cephydroxil (Cq30),
Chloramphenicol (C30), Cloxacillin (Cx30), Cephotaxime (Ce30),
Ceftazidime (Ca30), Ciprofloxacin (Cf5), Doxycycline Hydrochloride
(Do30), Gentamicin (G10), Metronidazole (Mt4), Neomycin (N30),
Norfloxacin (Nx10), Polymyxin B (Pb100), Rifampicin (R15),
Roxithromycin (Ro30), Tetracycline (T30), Trimethoprin (Tr30),
Vancomycin (Va30). The concentrations of the antibiotics in
microgram per disc were mentioned in parenthesis indicated above.
Mueller Hinton Agar medium (Himedia--M173) was used to grow each of
the microorganisms for antibiotic sensitivity test. The log phase
culture of each isolate was diluted 100 times and poured on Mueller
Hinton Agar (MHA) plates. It was swirled uniformly to distribute
the culture throughout the plate. After waiting for 30 minutes, the
cultures were carefully pipetted out from the plates and the plates
were allowed to dry. Then the antibiotic disks were placed onto the
MHA surface using the dispenser provided with the antibiotic discs.
The plates were incubated at 37.degree. C. for overnight. The zones
of inhibition were measured. The results were evaluated using
National Committee for Clinical Laboratory Standard's (NCCLS) chart
provided with the antibiotic discs by Himedia. The assay was done
thrice for each organism.
[0042] In some embodiments the protease is further characterized
by: a pH optimum in the range of 6-10.5; more than 90% proteolytic
activity at a temperature range of 4.degree. C. to 50.degree. C.;
minimum inhibition (0.4%) of proteolytic activity by 5 mM of Cu and
Co ions; inhibition of proteolytic activity (nearly 100%) by Cr and
Pb ions; and inhibition of activity by ethylenediaminetetraacetic
acid. The activity was measured as per the method of Malathu, R.,
et al., 2008 Characterization and Wash Performance Analysis of
Microbial Extracellular. American Journal of Applied Sciences, 5
(12): 16501661.)
[0043] The isolate was found to grow in the presence of heavy metal
salts like 1 mM NiCl.sub.2.6H.sub.2O, 3 mM CoCl.6H.sub.2O, 1 mM
Cr.sub.2O.sub.3, 2 mM CuSO.sub.4.5H.sub.2O, 2 mM Al
(NO.sub.3).sub.3.9H.sub.2O, 6 mM Pb(NO.sub.3), 0.2 mM HgCl.sub.2.
This strain was found to accumulate lead (1306 ppb), copper (132.42
ppb) and chromium (81.84 ppb) and distinct nano particles were
found in case of treatment with 5 ppm Pb, 0.5 ppm Cr, 2 ppm Cd, 0.1
ppm Hg and 1 ppm Cu. About 6-15 nano particles per cell were found
after Pb treatment and their size varied from 10-11 nm. In case of
Cr and Cu treatment, the elongation of cell size was observed which
can be one of the effects of metal induced stress on cell shape so
as to delay the cell division.
[0044] The protease content of cell free supernatant was determined
by a protease assay using hide powder azure as substrate as
discussed in Malathu, R., et al., 2008 Characterization and Wash
Performance Analysis of Microbial Extracellular. American Journal
of Applied Sciences, 5 (12): 1650-1661., which is hereby
incorporated by reference herein. The cell free supernatant was
used for enzyme quantification. Post overnight growth, the isolate
GZ was found to produce about 0.4 units of protease/ml, where 1
unit of activity can be defined as the amount of enzyme required to
produce an increase of 0.1 absorbance (OD at 440 nm). The protease
from GZ can tolerate a wide range of from pH 6-10.5 and was found
to retain 71% of its activity at pH 10.5 (FIG. 1a). The other
significant feature of the enzyme was its stability at high
temperatures up to 60.degree. C. with 74.4% activity retention
(FIG. 1b).
[0045] The protease activity was found to be more than 60% within
the range of pH 6-10.5 and the maximum activity was evident at pH
7.5. The method used was as reported by Malathu et al. Similarly,
the activity was demonstrated to be more than 90% within the range
of 4.degree. C. to 50.degree. C. and about 80% activity observed at
60.degree. C. Metal ions like Cu and Co exhibited minimum
inhibition on the enzyme activity whereas the presence of Cr and Pb
ions caused complete inhibition of enzyme activity. The
metalloprotease nature of the enzyme was confirmed by the
inhibition of activity by EDTA. Ionic detergent like SDS caused
minimal inhibition of the activity whereas Triton X-100 caused
complete inhibition. Agents like bleach, hydrogen peroxide and
beta-mercaptoethanol exert significant inhibition of enzyme
activity. There was complete inhibition of activity upon treatment
with PMSF and Leupeptin while partial inhibition in case of TPCK,
TLCK, Bestatin, EGTA, Ebalactone B and Phosphoramidone which
indicated the enzyme to be a serine protease. The enzyme was found
to possess the apr gene, the gene for alkaline protease.
Lipases
[0046] A particular lipase enzyme was isolated from a bacterial
culture (SRC_BS) obtained from Bheri (the shallow, flat bottomed,
waste water fed fish ponds) soil at East Calcutta Wetland
[22.degree.33' 12.06'' N and 88.degree.24' 41.05'' E]. Natural
remediation taking place in Bheri is further facilitated by the
alkaline pH generated by the addition of lime which in turn causes
decrease in coliform count from 23200 to 1090 (in raw sewage canal
and its corresponding Bheri). There is little change in
conductivity (1.6) and dissolved oxygen (3.8) among the two.
(Pradhan, A., P., et al., 2008. Phytoplankton diversity as
indicator of water quality for fish cultivation. American Journal
of Environmental Sciences, 4(4):271276.). It is a gram negative
short bacillus. The molecular identification of the isolate was
based on a partial sequence analysis of 16SrDNA, and the sequence
is available at GenBank as Acinetobacter sp. BS, with the accession
number FJ788517. This partial sequence shows 99% identity with
Acinetobacter sp HB1. The strain produces catalase and lipase and
is Vancomycin (30 .mu.g) and Ampicillin (10 .mu.g) resistant. The
strain does not secrete protease, oxidase, DNase, lecithinase or
amylase. The isolate was found to secrete 8 units/ml of lipase
after 16 hours of growth under optimum condition. Moreover the
isolate was found to grow in presence of heavy metal salts like 2
ppm NiCl.sub.2.6H.sub.2O, 1 ppm CoCl.6H.sub.2O, 2 ppm
Cr.sub.2O.sub.3, 2 ppm CuSO.sub.4.5H.sub.2O, 6 ppm Al
(NO.sub.3).sub.3.9H.sub.2O, 5 ppm Pb(NO.sub.3). The maximum
accumulation of accumulation was found with Pb salt treatment
(about 1446 ppb) followed by Cu at about 300 ppb. In comparison to
Pb and Cu, relatively less accumulation of lipase was found with Ni
(2.036 ppb), Co (2.293 ppb) and Cu. (9.253 ppb) treatment. Metals
like Ag were found to be localized inside and throughout the cell,
as determined through Transmission Electron Microscopy. This
isolate was also found to degrade crude oils like burnt mobil and
mobil. In liquid culture with 1% inoculum and 1.5% mobil, this
isolate demonstrated degradation efficiency of more than 70% and up
to 90% for different individual components of the mineral oil as
analyzed using GC-MS (Parkin Elmer XL with Flame ionization
detect). The details of the program used are as follows: Column
PE5, length 25 m, inner diameter 200 .mu.m, film thickness 0.33
.mu.M, Oven program including 4 min hold at 40.degree. C. with
ramping at 10.degree. C. per min up to 320.degree. C. followed by 8
min hold at 320.degree. C., total run time being 40 min. The
injector temperature was 200.degree. C., split flow at 25 ml/min,
helium as carrier at a flow rate of 0.7 ml/min)
[0047] Enzyme production was optimized in flask culture. About 5
units/ml of lipase enzyme was produced by the isolate BS after 16
hours of growth. One (1) unit of lipase enzyme was defined as
amount of enzyme releasing 1 umole of pNitrophenol per ml per min.
The cell free supernatant was used for enzyme assays. The enzyme
assay for lipase was done as reported by Sarkar et al., A. K., et
al., Microbial Biodiversity Screening from Mineral Ore Rich Site in
Andhra Pradesh, India, 2008. Online J. Biol. Sci. 8:32-40, which is
hereby incorporated by reference herein.
Amylases
[0048] Amylase enzyme was isolated from a bacterial culture
(SRC_SV1) obtained from the water of the marine coast of Vizag
under normal salinity [17.degree.43'04.69'' N and 83.degree.19'
54'' E (using GOOGLE EARTH)]. The molecular identification of the
isolate was based on a partial sequence analysis of 16SrDNA, and
the sequence is available at GenBank as Bacillus sp. SV1, with the
accession number FJ377723. This partial sequence shows 99.91%
identity with Bacillus cibi. The strain shows resistance to
Metronidazole.
[0049] FIG. 9 depicts the results of an assay of amylase activity
on starch residue on a plate using Himedia starch agar as per the
manufacturer's instructions (HIMEDIA LABORATORIES. PVT. LTD.). The
extracellular supernatant was used as the source of amylase.
Enzyme Production
[0050] Enzymatic detergent additive-producing strains may be used
to secrete the additives by any available methods, including, for
example, by batch fermentation and by immobilization of the
microbes. Fermentation can be conducted and controlled by various
techniques. Appropriate nutrient cultures may be prepared,
including, for example, carbon and nitrogen sources and other
nutritional substances that favor or are necessary for the growth
of the particular microbes (e.g., bacteria). Sugars and
sugar-containing substances may be included as suitable sources of
carbon, including, for example, but not limited to, starch,
dextrin, cane sugar, lactose, maltose, fructose, and glucose.
Nitrogen sources include, for example, protein-containing
substances, such as peptone from soy beans, meat, casein, gelatin,
yeast protein or yeast extract, wastes from the processing of meat
or animal bodies, and ammonium salts. Other examples of nutrients
include, for example, inorganic salts, for exampled alkaline and
alkali earth metal salts and phosphates, together with trace
elements, such as, for example, Fe, Mg, Mn, Co, and Ni.
[0051] Fermentation may be carried out at appropriate pH levels and
temperatures to maximize microbe growth and secretion of enzyme,
including, for example, at pH levels between about 5 and 9, or, for
example, at pH levels between about 6 and 8. The temperature may
be, for example, between about 33 to about 45 degrees Celsius, or,
for example, between about 35 to about 39 degrees Celsius, or, for
example, at about 37 degrees Celsius.
[0052] Enzyme may also be produced using immobilized microbial
cells, for example, as presented in Kumar, S. R. and M.
Chandrasekharan, 2003. Continuous production of L-glutaminase by an
immobilized marine Pseudomonas sp BTMS-51 in a packed bed reactor.
Process Biochem., 38: 1431-1436.
[0053] The cells were immobilized both in alginate beads as well as
on boiled straw packed in plastic perforated containers which were
in turn placed inside bigger containers with medium for microbial
growth.
Enzyme Additive Formulation
[0054] Enzyme additives may be provided alone, or in detergent
compositions. For example, protease, lipase, or amylase may each be
formulated as separate additive compositions, including, for
exampled, stabilizers or fillers. These compositions may be
provided in liquid, powder, or granulate form. The enzyme additives
may also, for example, include the enzymes in combination, for
example, protease and lipase, protease and amylase, amylase and
lipase, or protease, lipase and amylase. These additives may also
include other enzyme additives. Detergent compositions may comprise
the enzyme additives. The enzyme additives may further be provided
in the form of a kit, including instructions regarding, for
example, the use of the additives to remove stains, recommended
washing conditions, or the proportions of additive and detergent to
be used. The enzyme additive may be in a container, such as, for
example, a tube, dissolvable packet, vial, pouch, or other
container, and may, for example, be in a powder, granular, liquid,
or other form. Kits may include more than one enzyme additive, for
example, the kit may include both protease and lipase additives,
and the protease and lipase additives may be provided in separate
containers, or may be provided in the same container. The kit may
further provide a detergent composition.
EXAMPLES
[0055] The examples set forth below illustrate certain embodiments
and do not limit the disclosed technology.
Example 1
Proteases as Detergent Additives
[0056] Protease isolated from strain SRC_GZ, added to detergent,
enhanced stain removal. The protease was mixed with standard
detergent, including Blue Bird detergent as well as others noted
herein. at a proportion of 6 units of enzyme/gm of detergent. Cloth
pieces of 1.5.times.1.5 inches were stained with grease car door,
car battery, and/or car engine grease, and kept for an hour before
incubation with detergent. The cloth pieces were dipped in
different combinations as stated: 1) only water (considered as
negative control), 2) only detergent, 3) detergent enzyme mixture
and 4) only enzyme. In the graph, Prot stands for Protease, Det
stands for Detergent while Det+Prot stands for detergent with
protease. The washing efficiency was evaluated after one hour of
treatment by observing the stain removal after simple rubbing and
rinsing with water. The extent of stain removal was analyzed by
densitometric scanning of the residual stain on the cloth using
software Quantity 1 from BIORAD. The protease treatment was found
to have a better cleaning efficiency (70% stain removal) than just
detergent (68% stain removal) but the best efficiency was obtained
on mixing both the detergent and the protease (74% stain removal)
(FIG. 2).
[0057] The protease additive was compatible with different
detergents. Protease enzyme at the same concentration (6 unit/gm of
detergent) was mixed with different detergents available in
commercial markets, namely SURF, EXCEL (Hindustan Lever Limited,
Mumbai, India), TIDE (Procter and Gamble, USA), AERIAL (Procter and
Gamble, USA), SUNLIGHT (Unilever, South Africa), NIRMA (Nirma Ltd.,
Ahmedabad, India), SAGAR (AMOCHEM, Kolkata, India), BLUE BIRD
(Local Make, Kolkata, India), JET (Hindusthan Chemical Company,
Kolkata, India), SODA (local make, Kolkata, India), and VIM
(Hindusthan Lever Limited, India). FIG. 3 shows the enhancement of
cleaning efficiency when adding the protease to various detergents.
The stain density before washing was considered as 100%, and after
washing, the remaining stain was compared for the various
detergents, with or without the protease enzyme additive.
[0058] The effect of the enzyme as a detergent additive on various
stains on different fabrics such as cotton, silk, chiffons, and
synthetic material was assayed, with the results shown in FIG. 4.
The x-axis represents the different fabrics used while y-axis
represents the percent of residual stain post wash as compared to
just stained cloth taken as 100%. FIG. 5 demonstrates that the
protease addition increased the stain removal efficiency for
removing stains like grease, burnt mobile, vegetable curry and
blood. FIG. 5 represents the effect of the protease additive on the
cleaning efficiency of various stains as obtained by densitometric
scanning. The x-axis shows the different sources of stain and the
y-axis represents the percent of residual stain post wash as
compared to unwashed stain, considered as 100%.
[0059] The intact activity of the protease enzyme as a detergent
additive was found for a period of two months (FIG. 6a, 6b). The
residual activity could be traced back after more than a year.
Example 2
Repeated Cycling of Immobilized Cells for Enzyme Production
[0060] Immobilized SRC_GZ bacteria was used to produce protease
using methods essentially as presented in Kumar, S. R. and M.
Chandrasekharan, 2003. Continuous production of L-glutaminase by an
immobilized marine Pseudomonas sp BTMS-51 in a packed bed reactor.
Process Biochem., 38: 1431-1436. The protease-producing cells were
immobilized in 8% Na-alginate solution and 1M CaCl.sub.2. The
enzyme produced upon immobilization of cells (1% inoculum)
demonstrated a higher efficiency when used to clean stains when in
a shaking condition (1 U/ml activity) when the supernatant was
compared to a stationary condition (0.5 U/ml activity). The enzyme
production was relatively stable throughout the 20 cycles both for
static and shaking conditions. (FIG. 7).
Example 3
Lipase Increases the Efficiency of Detergent Additives Including
Protease
[0061] Adding bacterial lipase to detergent and protease additive
increases the efficiency of stain removal. Lipase production was
done under shake flask conditions at 37.degree. C. with continuous
shaking at 150 rpm. The standard combination was addition of 3 ml
(24 units) of extracellular supernatant containing lipase to 0.25
gm of detergent containing 6 units of protease/gm of detergent. A
market survey was conducted to determine the acceptability of the
formulated detergent. As shown in FIG. 8, the formulated detergent
was acceptable to the survey participants. The sample size for the
three separate survey were 34, 37, and 31 families respectively
from different strata of the society) There was 90% acceptance for
use in washing cloths, 96.6% for washing steel utensil, 95.23%
accepted the formulation for washing kitchens, 96.42% for washing
glassware, 95.5% for washing floors, 90% accepted for cleaning
commode/tiles and 94.4% accepted the detergent formulation for
washing sink and basins (FIG. 8a-f).
Example 4
Amylase Increases the Efficiency of Detergent Additives Including
Protease and Lipase
[0062] Adding bacterial amylase to detergent, supplemented with
protease and lipase additives, further increases the efficiency of
stain removal.
[0063] 3 ml of cell-free SRC-SV1 supernatant was added to 0.25 gm
of detergent containing lipase (24 units) and protease (6
units/gm). It enhanced the cleaning efficiency several fold, as
shown in FIG. 10. The top panel represents the following from left
to right: the negative control represents stain washed in water;
positive control was washed with 0.25 gm of detergent; washed with
supernatant from the fourth recharge of the packed bed bioreactor.
The bottom panel from left to right were as follows: washed with
supernatant from the 11.sup.th recharge; washed with supernatant
from the 11.sup.th recharge along with 3 ml of amylase; washed with
supernatant from the 12.sup.th recharge; and washed with
supernatant from the 12.sup.th recharge along with 3 ml of
amylase.
Example 5
16SrDNA Sequences of Bacteria Used for Isolation and Identification
of Enzymes
[0064] This example provides 16SrDNA sequences of protease, lipase,
and amylase, as deposited in GenBank.
TABLE-US-00001 SEQ ID NO: 1 Microbacterium SRC 010 (Deposit
Accession number MTCC 5565) 16S ribosomal RNA gene, partial
sequence 1 ggatgaacgc tggcggcgtg cttaacacat gcaagtcgaa cggtgaagca
ggagcttgct 61 cttgtggatc agtggcgaac gggtgagtaa cacgtgagca
acctgcccct gactctggga 121 taagcgctgg aaacggcgtc taatactgga
tatgtgacgt gaccgcatgg tctgcgtttg 181 gaaagatttt tcggttgggg
atgggctcgc ggcctatcag cttgttggtg aggtaatggc 241 tcaccaaggc
gtcgacgggt agccggcctg agagggtgac cggccacact gggactgaga 301
cacggcccag actcctacgg gaggcagcag tggggaatat tgcacaatgg gcgaaagcct
361 gatgcagcaa cgccgcgtga gggatgacgg ccttcgggtt gtaaacctct
tttagcaggg 421 aagaagcgaa agtgacggta cctgcagaaa aagcgccggc
taactacgt SEQ ID NO: 2 Acinetobacter sp. BS (Deposit Accession
number MTCC 5563) 16S ribosomal RNA gene partial sequence 1
tagagtttga tcatggctca gattgaacgc tggcggcagg cttaacacat gcaagtcgag
61 cgggggaagt agcttgctac tggacctagc ggcggacggg tgagtaatgc
ttaggaatct 121 gcctattagt gggggacaac attccgaaag gaatgctaat
accgcatacg tcctacggga 181 gaaagcaggg gaccttcggg ccttgcgcta
atagatgagc ctaagtcgga ttagctagtt 241 ggtggggtaa aggcctacca
aggcgacgat ctgtagcggg tctgagagga tgatccgcca 301 cactgggact
gagacacggc ccagactcct acgggaggca gcagtgggga atattggaca 361
atggggggaa ccctgatcca gccatgccgc gtgtgtgaag aaggccttat ggttgtaaag
421 cactttaagc gaggaggagg ctactagtat taatactact ggatagtgga
cgttactcgc 481 agaataagca ccggctaact ctgtgccagc agccgcggta
atacagaggg tgcgagcgtt 541 aatcggattt actgggcgta aagcgtgcgt
aggcggccat ttaagtcaaa tgtgaaatcc 601 ccgagcttaa cttgggaatt
gcattcgata ctggatggct agagtatggg agaggatggt 661 agaattccag
gtgtagcggt gaaatgcgta gagatctgga ggaataccga tggcgaaggc 721 agccatct
SEQ ID NO: 3 Bacillus SV-1 (Deposit Accession number MTCC 5567) 16S
ribosomal RNA gene partial sequence 1 ggacgaacgc tggcggcgtg
cctaatacat gcaagtcgag cggayctctt cggagrtcag 61 cggcggacgg
gtgagtaaca cgtgggcaac ctgcctgtaa gactgggata actccgggaa 121
accggagcta ataccggata ctatgtcaaa ccgcatggtt tgacattcaa agacggtttc
181 ggctgtcact tacagatggg cccgcggcgc attagctagt tggtgaggta
atggctcacc 241 aaggcgacga tgcgtagccg acctgagagg gtgatcggcc
acactgggac tgagacacgg 301 cccagactcc tacgggaggc agcagtaggg
aatcttccgc aatggacgaa agtctgacgg 361 agcaacgccg cgtgagtgat
gaaggttttc ggatcgtaaa actctgttgt cagggaagaa 421 caagtgccgg
agtaactgcc ggcgccttga cggtacctga ccagaaagcc acggctaact 481 ac
[0065] The entirety of each patent, patent application, publication
and document referenced herein hereby is incorporated by reference.
Citation of the above patents, patent applications, publications
and documents is not an admission that any of the foregoing is
pertinent prior art, nor does it constitute any admission as to the
contents or date of these publications or documents.
[0066] The present disclosure is not to be limited in terms of
particular embodiments described in this disclosure, which are
illustrations of various aspects. Many modifications and variations
can be made without departing from the spirit and scope of the
disclosure, as will be apparent to those skilled in the art.
Functionally equivalent methods and apparatuses within the scope of
the disclosure, in addition to those enumerated herein, will be
apparent to those skilled in the art from the foregoing
descriptions. Such modifications and variations fall within the
scope of the appended claims. The present disclosure is to be
limited only by the terms of claims (e.g., the claims appended
hereto) along with the full scope of equivalents to which such
claims are entitled. It is to be understood that this disclosure is
not limited to particular methods, reagents, compounds compositions
or biological systems, which can, of course, vary. It is also to be
understood that terminology used herein is for the purpose of
describing particular embodiments only, and is not necessarily
limiting.
[0067] With respect to the use of substantially any plural and/or
singular terms herein, those having skill in the art can translate
from the plural to the singular and/or from the singular to the
plural as is appropriate to the context and/or application. Various
singular/plural permutations may be expressly set forth herein for
sake of clarity.
[0068] It will be understood by those within the art that, in
general, terms used herein, and especially in the appended claims
(e.g., bodies of the appended claims) are generally intended as
"open" terms (e.g., the term "including" should be interpreted as
"including but not limited to," the term "having" should be
interpreted as "having at least," the term "includes" should be
interpreted as "includes but is not limited to," etc.). It will be
further understood by those within the art that if a specific
number of an introduced claim recitation is intended, such an
intent will be explicitly recited in the claim, and in the absence
of such recitation no such intent is present. For example, as an
aid to understanding, the following appended claims may contain
usage of the introductory phrases "at least one" and "one or more"
to introduce claim recitations. However, the use of such phrases
should not be construed to imply that the introduction of a claim
recitation by the indefinite articles "a" or "an" limits any
particular claim containing such introduced claim recitation to
embodiments containing only one such recitation, even when the same
claim includes the introductory phrases "one or more" or "at least
one" and indefinite articles such as "a" or "an" (e.g., "a" and/or
"an" should be interpreted to mean "at least one" or "one or
more"); the same holds true for the use of definite articles used
to introduce claim recitations. In addition, even if a specific
number of an introduced claim recitation is explicitly recited,
those skilled in the art will recognize that such recitation should
be interpreted to mean at least the recited number (e.g., the bare
recitation of "two recitations," without other modifiers, means at
least two recitations, or two or more recitations).
[0069] Furthermore, in those instances where a convention analogous
to "at least one of A, B, and C, etc." is used, in general such a
construction is intended in the sense one having skill in the art
would understand the convention (e.g., "a system having at least
one of A, B, and C" would include but not be limited to systems
that have A alone, B alone, C alone, A and B together, A and C
together, B and C together, and/or A, B, and C together, etc.). In
those instances where a convention analogous to "at least one of A,
B, or C, etc." is used, in general such a construction is intended
in the sense one having skill in the art would understand the
convention (e.g., "a system having at least one of A, B, or C"
would include but not be limited to systems that have A alone, B
alone, C alone, A and B together, A and C together, B and C
together, and/or A, B, and C together, etc.).
[0070] The term "about" as used herein refers to a value within 10%
of the underlying parameter (i.e., plus or minus 10%), and use of
the term "about" at the beginning of a string of values modifies
each of the values (i.e., "about 1, 2 and 3" refers to about 1,
about 2 and about 3). For example, a weight of "about 100 grams"
can include weights between 90 grams and 110 grams. Further, when a
listing of values is described herein (e.g., about 50%, 60%, 70%,
80%, 85% or 86%) the listing includes all intermediate and
fractional values thereof (e.g., 54%, 85.4%). It will be further
understood by those within the art that virtually any disjunctive
word and/or phrase presenting two or more alternative terms,
whether in the description, claims, or drawings, should be
understood to contemplate the possibilities of including one of the
terms, either of the terms, or both terms. For example, the phrase
"A or B" will be understood to include the possibilities of "A" or
"B" or "A and B."
[0071] In addition, where features or aspects of the disclosure are
described in terms of Markush groups, those skilled in the art will
recognize that the disclosure is also thereby described in terms of
any individual member or subgroup of members of the Markush
group.
[0072] Thus, it should be understood that although the present
technology has been specifically disclosed by representative
embodiments and optional features, modification and variation of
the concepts herein disclosed may be resorted to by those skilled
in the art, and such modifications and variations are considered
within the scope of this technology. As will be understood by one
skilled in the art, for any and all purposes, such as in terms of
providing a written description, all ranges disclosed herein also
encompass any and all possible subranges and combinations of
subranges thereof. Any listed range can be easily recognized as
sufficiently describing and enabling the same range being broken
down into at least equal halves, thirds, quarters, fifths, tenths,
etc. As a non-limiting example, each range discussed herein can be
readily broken down into a lower third, middle third and upper
third, etc. As will also be understood by one skilled in the art
all language such as "up to," "at least," "greater than," "less
than," and the like include the number recited and refer to ranges
which can be subsequently broken down into subranges as discussed
above. Finally, as will be understood by one skilled in the art, a
range includes each individual member. Thus, for example, a group
having 1-3 cells refers to groups having 1, 2, or 3 cells.
Similarly, a group having 1-5 cells refers to groups having 1, 2,
3, 4, or 5 cells, and so forth.
[0073] While various aspects and embodiments have been disclosed
herein, other aspects and embodiments will be apparent to those
skilled in the art. The various aspects and embodiments disclosed
herein are for purposes of illustration and are not limiting, with
the true scope and spirit of certain embodiments indicated by the
following claims.
Sequence CWU 1
1
31469DNAMicrobacterium sp. 1ggatgaacgc tggcggcgtg cttaacacat
gcaagtcgaa cggtgaagca ggagcttgct 60cttgtggatc agtggcgaac gggtgagtaa
cacgtgagca acctgcccct gactctggga 120taagcgctgg aaacggcgtc
taatactgga tatgtgacgt gaccgcatgg tctgcgtttg 180gaaagatttt
tcggttgggg atgggctcgc ggcctatcag cttgttggtg aggtaatggc
240tcaccaaggc gtcgacgggt agccggcctg agagggtgac cggccacact
gggactgaga 300cacggcccag actcctacgg gaggcagcag tggggaatat
tgcacaatgg gcgaaagcct 360gatgcagcaa cgccgcgtga gggatgacgg
ccttcgggtt gtaaacctct tttagcaggg 420aagaagcgaa agtgacggta
cctgcagaaa aagcgccggc taactacgt 4692728DNAAcinetobacter sp.
2tagagtttga tcatggctca gattgaacgc tggcggcagg cttaacacat gcaagtcgag
60cgggggaagt agcttgctac tggacctagc ggcggacggg tgagtaatgc ttaggaatct
120gcctattagt gggggacaac attccgaaag gaatgctaat accgcatacg
tcctacggga 180gaaagcaggg gaccttcggg ccttgcgcta atagatgagc
ctaagtcgga ttagctagtt 240ggtggggtaa aggcctacca aggcgacgat
ctgtagcggg tctgagagga tgatccgcca 300cactgggact gagacacggc
ccagactcct acgggaggca gcagtgggga atattggaca 360atggggggaa
ccctgatcca gccatgccgc gtgtgtgaag aaggccttat ggttgtaaag
420cactttaagc gaggaggagg ctactagtat taatactact ggatagtgga
cgttactcgc 480agaataagca ccggctaact ctgtgccagc agccgcggta
atacagaggg tgcgagcgtt 540aatcggattt actgggcgta aagcgtgcgt
aggcggccat ttaagtcaaa tgtgaaatcc 600ccgagcttaa cttgggaatt
gcattcgata ctggatggct agagtatggg agaggatggt 660agaattccag
gtgtagcggt gaaatgcgta gagatctgga ggaataccga tggcgaaggc 720agccatct
7283482DNABacillus sp. 3ggacgaacgc tggcggcgtg cctaatacat gcaagtcgag
cggayctctt cggagrtcag 60cggcggacgg gtgagtaaca cgtgggcaac ctgcctgtaa
gactgggata actccgggaa 120accggagcta ataccggata ctatgtcaaa
ccgcatggtt tgacattcaa agacggtttc 180ggctgtcact tacagatggg
cccgcggcgc attagctagt tggtgaggta atggctcacc 240aaggcgacga
tgcgtagccg acctgagagg gtgatcggcc acactgggac tgagacacgg
300cccagactcc tacgggaggc agcagtaggg aatcttccgc aatggacgaa
agtctgacgg 360agcaacgccg cgtgagtgat gaaggttttc ggatcgtaaa
actctgttgt cagggaagaa 420caagtgccgg agtaactgcc ggcgccttga
cggtacctga ccagaaagcc acggctaact 480ac 482
* * * * *